1     Analysis of the crystallization process of a 
           2     biopharmaceutical compound in the presence of 
           3     impurities using process analytical technology 
           4     (PAT) tools 
                                1             2,3                   1,3
           5     Elena Simone,  Wei Zhang,   Zoltan K. Nagy*   
           6      
           7     1 Department of Chemical Engineering, Loughborough University, Loughborough, LE11 
           8     3TU, U.K. 
           9     2 New drug research and development center, North China Pharmaceutical group 
          10     Corporation, Shijiazhuang, China  
          11     3 School of Chemical Engineering, Purdue University, West Lafayette, 47907, Indiana, 
          12     United State 
          13                                      
                                                                  1 
                  
           1     Abstract 
           2     The crystallization of biopharmaceuticals can be problematic since, because the biosynthesis 
           3     of these compounds is very difficult to control, they can present a significant amount of 
           4     impurities that has to be eliminated. In fact, impurities can lead to changes in the properties of 
           5     the drug that can significantly reduce its effectiveness or even put in danger the user.  
           6     The substance used in this work is vitamin B12 crude product extracted from fermentation. 
           7     The aim of this work is to exploit process analytical technology (PAT) tools to study the 
           8     crystallization step of vitamin B12.  Linear cooling crystallization experiments were 
           9     performed using different conditions. The effects of solvent, cooling rate, seeding and purity 
          10     of the initial material on the final size distribution and purity of the crystals were investigated 
          11     through the use of UV/Vis spectroscopy, focused beam reflectance measurement (FBRM) 
          12     and the CryPRINS software (Crystallization Process Informatics System). 
          13     It was found that impurities strongly inhibit the growth of vitamin B12 crystals, promoting 
          14     nucleation and leading to a poor final crystal size distribution. Slow cooling can help in 
          15     increasing the purity of the final product but also generates a broad crystal size distribution 
          16     because of secondary nucleation. Preparing the solution with material already crystallized 
          17     once and using purified seeds helped in obtaining a narrower crystal size distribution and also 
          18     reduces breakage. 
          19      
          20     Keywords: PAT tools, biopharmaceutical, vitamin, crystallization, monitoring 
          21                                      
                                                                  2 
                  
           1     Introduction 
           2     Over the last decade the investment in research and development of biopharmaceutical 
           3     companies has more than doubled: there is a great interest in formulating and optimizing the 
           4     manufacturing of bio-molecules and biopharmaceuticals such as proteins, lipids, vitamins, 
           5     hormones and DNA. Those compounds can be used in the treatment of serious diseases or 
           6     just as nutritional supplement.  
           7     Vitamin B12 (cobalamin) is one of the biggest and most complex vitamins and, in humans, it 
           8     is required to assist the actions of two enzymes: methionine synthase and (R)-methylmalonyl-
           9     CoA mutase. It is commonly used to cure pernicious anemia and its deficiency determines an 
          10     increase in homocysteine levels that leads to a major  risk for heart disease, stroke, 
          11     atherosclerosis and vascular diseases. Despite the lack of solid evidences on the importance 
          12     of vitamin B12 in maintaining normal myelination of nerve cells, its role in preventing many 
          13     neurological and psychiatric symptoms is becoming clearer and clearer. For these reasons, 
          14     every year, around 10 tons of the semisynthetic version of vitamin B12 (cyanocobalamin) are 
                                                                                 1
          15     produced by biofermentation from several bacterial species.   
          16     A schematic of vitamin B12 molecule is shown in Figure 1. The molecule is fairly large and 
          17     complex and can be divided in three parts: (1) a central corrin ring which contains four 
          18     ligands for the central cobalt ion, (2) a lower ligand donated by the 5,6-
          19     dimethylbenzimidazole (DMBI) and (3) an upper ligand made from either an adenosyl group 
          20     or a methyl group. 
          21     Two pseudo-polymorphic  forms were discovered by X-ray diffraction: a wet and a  dry 
                                       2,3
          22     crystalline structure.    The main difference between the two is the presence of water 
          23     molecules in the crystal lattice that makes the unit cell of the wet form bigger than the dry. 
          24     Cyanocobalamin is mainly produced by biosynthetic fermentation processes, although a full 
                                                                                                   1
          25     chemical synthesis (consisting of 70 steps) was developed in the early 70s.   High levels of 
                                                                  3 
                  
           1     impurities are present in the biosynthesized vitamin B12 that can inhibit growth  of the 
           2     crystals  and strongly affect the final crystal size distribution  together with the ease of 
           3     downstream processes (filtration in particular).  
           4     Process analytical technology (PAT)  tools are widely used for the development of the 
                                                                                   4-6
           5     crystallization processes of many synthetic pharmaceuticals.        . The term PAT  refers to “a 
           6     system for designing, analyzing, and controlling manufacturing trough timely measurement 
           7     of critical quality and performance attributes of raw and in-process material and processes, 
                                                                    7
           8     with the goal of ensuring final product quality”.   PAT tools are the main element used in the 
           9     “Quality by Design” (QbD) approach that was introduced in the pharmaceutical industries in 
          10     order to minimize product waste due to mistakes in the manufacturing process. The QbD 
          11     concept consists in obtaining the desired quality of the product though the correct design of 
          12     the manufacturing process. In this approach some critical variables of the process, strictly 
          13     related to the quality of the product, are controlled during the manufacturing in order to 
                                                            8,9
          14     control the quality of the product itself.     The QbD approach is well known and applied in 
          15     the pharmaceutical industry but only few examples are present for the manufacturing of 
                                      10,11
          16     biopharmaceuticals.        Despite the difficulty of controlling biological processes  Near 
          17     infrared (NIR) and Raman spectroscopy, HPLC and image analysis were recently used for 
                                                               12-14
          18     some manufacturing steps of biomolecules.           
          19     In this work the crystallization of vitamin B12 will be studied with PAT tools (ATR-UV/Vis, 
          20     FBRM and CryPRINS) and the effect of impurities on the growth and nucleation rate will be 
          21     analyzed. The produced crystals are analyzed with optical microscopy, HPLC and Raman. 
          22     Most of the impurities in cyanocobalamin have high levels of fluorescence and, therefore, 
          23     Raman can give an indication of the purity of the crystals since it is highly sensitive to this 
                               15,16
          24     phenomenon.        However, for a precise quantification of the type and level of impurity in 
          25     vitamin B12 samples HPLC is necessary. The paper provides one of the first comprehensive 
                                                                  4